Plant, Animal & Microbe Genomes X Conference

January 12-16, 2002
Town & Country Convention Center
San Diego, CA

Workshop: Compositae
            

MAXIMIZING GENE DISCOVERY IN LETTUCE AND SUNFLOWER

¹ Celera AgGen, 1756 Picasso Avenue, Davis, CA 95616 USA

² Department of Vegetable Crops, University of California, Davis, CA 95616 USA

³ Department of Crop and Soil Science, Oregon State University, Corvallis, CA 97331 USA

⁴ Intellectual Force, 2604 Kalamazoo Place, Davis, CA 95616 USA

A comprehensive annotated EST database is a valuable resource for gene discovery and comparative genomics. One of the goals of the Composite Genomics Project (CGP) is to contribute as many annotated EST sequences from lettuce and sunflower as possible to public databases. To generate this resource in a cost effective and timely manner, the CGP collaborated with Celera to couple customized library construction with high throughput sequencing and bioinformatics. RNA was extracted from two parental genotypes of a core mapping population for each species to increase the chances of identifying SNPs in silico. Multiple tissues and treatments were included to enhance the diversity of transcripts. Tissue/treatment specific sequence tags were incorporated into the primers used for the production of cDNA to allow the source of ESTs to be determined. cDNAs were size selected and pooled into four fractions and four size selected libraries were constructed for each genotype to minimize size bias during cloning and transformation. The combination of tissue/treatment tagging and size fractionation allowed us to pool tissues into a limited number of libraries per genotype for efficient high throughput sequencing. QC sequencing was performed to assess the quality and diversity of the libraries and to determine the composition of the libraries submitted for generation of approximately 50,000 reads per species. The effectiveness of this strategy in identifying a broad spectrum of genes will be presented.